A Study On The Relationship Between Surface Electrostatics And Conformational Flexibility Of Psychrophilic Serine Proteases

Psychrophilic enzymes has the high catalytic activity at low temperatures which in turn guarantees the normal metabolism rate of the psychrophiles.Probing the molecular mechanisms by which the psychrophilic enzymes adapt to the low-temperature condition has become the core topic in the fields of enzymology and protein engineering.Numerous studies on the cold adaptation of the psychrophilic enzyme have shown that the high conformational flexibility of enzyme structure is a fundamental basis for maintaining the sufficiently high catalytic activity of an enzyme under the low temperature;however,the questions of which physicochemical properties of the enzyme and what factors affect the conformational flexibility of the enzyme remain still unclear.In this thesis,the orthologous memebers of differently temperature-adapted enzymes from the subtilisin-like serine protease family,i.e.,the psychrophilic VPR and mesophilic PRK,were used as research objects to investigate the relationship between the surface electrostatic properties and the flexibility of enzyme structure using molecular simulations combined with electrostatic calculation method.In this thesis,the structures of these two proteases were subjected to multiple-replica molecular dynamics(MD)simulations(10×100 ns),respectively,based on which the concatenated equilibrium trajectories were obtained and used for calculating the geometrical properties of two serine proteases,including the total solvent accessible surface area(SASA),relative solvent accessible surface(R-SASA)of each amino acid residue,per-residue Cα root mean square fluctuation(RMSF),and the number of hydrogen bonds formed between protein and solvent(including numbers of both the dynamic hydrogen bonds and the static hydrogen bonds).In addition,through solving Poisson-Boltzmann equation with finite difference approximation method,the electrostatic surface potential were calculated on the representative structures of the two proteases which were obtained from clustering structural snapshots of the concatenated equilibrium trajectories of the two proteases.Finally,the pair-wise pearson correlation coefficients among all structural parameters were calculated for these two proteases,and the linear regression analyses were performed to interpret the extent to which a variety of structural parameters would influence the variation of Ca RMSF value.Comparison between Cα RMSF values of the amino acids at the structural equivalent position of these two proteases reveals that most regions including the peripheral loops,substrate-binding regions,and the C-terminus have higher flexibility in VPR than in PRK,which seems likely to be a major cause for maintenance of high catalytic activity of the cold-adapted VPR at low temperatures.Comparative analysis of geometric properties reveals that,when compared to PRK,VPR has larger solvent accessible surface area(SASA),more number of dynamic hydrogen bonds(NDHB)formed between protein and solvent,and a slightly higher number of intermolecular static hydrogen bonds(NSHB).Comparison between the electrostatic surface potential of two proteases reveals that although differently charged electrostatic potential patches(such as electropositive,electronegative,and electroneutral patches)promiscuously spread over the front surfaces of the two proteases,distinctly different distributions of electrostatic potentials can be observed on the back surfaces of the two proteases:the back surface of VPR is dominated by the electronegative potential,while that of PRK is largely covered by the electropositive potential,We speculate that the difference in distributions of differently charged electrostatic potentials on the two peotease surfaces could regulate/affect the local flexibility/rigidity of proteases.In order to validate this speculation,we first classified all surface residues into three groups,i.e.,the electronegative potential residues,electropostive potential residues,and electroneutral potential residues according to whether they are covered by electronegative,electropositive,or electroneutral potentials,and then performed a series of statistical analysis at the residue level on the Ca RMSF values and numbers of protein-solvent dynamic/static hydrogen bonds.The results show that the electronegative potential residues have higher conformational flexibility and more NSHB than the electropostive potential residues and electroneutral potential residues,evidencing that the stable(static)hydrogen-bonding interactions between the electronegative potential residues and solvent contribute to enhancing flexibility of the electronegative potential surface.The results of pairwise correlation analysis among all structural parameters show that,for both proteases,R-SASA has high correlation with all the other eight parameters,and RMSF has the greatest correlation with R-SASA,negatively charged surface area(NeSA),and NDHB of the residue main chain(MC-NDHB).The results of linear regression analysis reveal that for both PRK and VPR,the independent variables R-SASA,NeSA,and MC-NDHB can account for the highest proportion of variance(R2)in the dependent variable RMSF,and this is consistent with the results of correlation analysis,both implying that the favorable hydrogen-bonding interactions between the highly exposed main-chain polar atoms with negative partial charges and the solvent can contribute to enhancing the local flexibility of the negatively charged protein surface.Taken together,the surface electrostatic properties are closely related to the protein conformational flexibility:the surface electronegativity can greatly contribute to promoting the protein conformational flexibility while the surface electropositivitv and electroneutrality make very limited contribution to conformational flexibility or even contribute to enhancing the conformational rigidity.When compared to the electropositive or electroneutral surface,the negatively charged surface can form more favorable and stable hydrogen-bonding interactions with water molecules,and this is likely to be a very crucial factor responsible for the observed higher flexibility of the negatively charged protein surface.When compared to the mesophilic serine protease PRK,for the psychrophilic VPR,the greater exposure of the negatively charged surface arising from its more negatively charged residues and polar uncharged residues is the primary reason for its increased conformational flexibility.The results of this thesis would facilitate an in-depth understanding of the origin of protein conformational flexibility and of the cold-adaptation mechanisms of psychrophilic enzymes.